Transcriptional regulation of flavonoid biosynthesis in nectarine (Prunus persica) by a set of R2R3 MYB transcription factors

Abstract

Background: Flavonoids such as anthocyanins, flavonols and proanthocyanidins, play a central role in fruit colour, flavour and health attributes. In peach and nectarine (Prunus persica) these compounds vary during fruit growth and ripening. Flavonoids are produced by a well studied pathway which is transcriptionally regulated by members of the MYB and bHLH transcription factor families. We have isolated nectarine flavonoid regulating genes and examined their expression patterns, which suggests a critical role in the regulation of flavonoid biosynthesis.

Results: In nectarine, expression of the genes encoding enzymes of the flavonoid pathway correlated with the concentration of proanthocyanidins, which strongly increases at mid-development. In contrast, the only gene which showed a similar pattern to anthocyanin concentration was UDP-glucose-flavonoid-3-O-glucosyltransferase (UFGT), which was high at the beginning and end of fruit growth, remaining low during the other developmental stages. Expression of flavonol synthase (FLS1) correlated with flavonol levels, both temporally and in a tissue specific manner. The pattern of UFGT gene expression may be explained by the involvement of different transcription factors, which up-regulate flavonoid biosynthesis (MYB10, MYB123, and bHLH3), or repress (MYB111 and MYB16) the transcription of the biosynthetic genes. The expression of a potential proanthocyanidin-regulating transcription factor, MYBPA1, corresponded with proanthocyanidin levels. Functional assays of these transcription factors were used to test the specificity for flavonoid regulation.

Conclusions: MYB10 positively regulates the promoters of UFGT and dihydroflavonol 4-reductase (DFR) but not leucoanthocyanidin reductase (LAR). In contrast, MYBPA1 trans-activates the promoters of DFR and LAR, but not UFGT. This suggests exclusive roles of anthocyanin regulation by MYB10 and proanthocyanidin regulation by MYBPA1. Further, these transcription factors appeared to be responsive to both developmental and environmental stimuli.

Figure 1

Scheme of the flavonoid biosynthetic pathway in plants. Genes encoding enzymes for each step are indicated as follows: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol 4-reductase; LDOX, leucoanthocyanidin dioxygenase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; UFGT, UDP-glucose:flavonoid-3-O-glycosyltransferase.

Figure 2

Fruit development series of ‘Stark Red Gold’ nectarines (A) and their anthocyanin (B) and proanthocyanidin (C) and (D) flavonol concentration (mg g^-1 dry weight). The developmental stages are indicated as follows: S1, 50 DAFB; S2, 66 DAFB; S2-3, 74 DAFB; S3, 90 DAFB; S4, 135 DAFB. Error bars are standard deviation of the mean for five replicates.

Figure 3

Relative expression profiling of biosynthetic genes for main pathway flavonoid and anthocyanin synthesis in skin and flesh of ‘Stark Red Gold’ nectarines. The developmental stages are indicated as follows: S1, 50 DAFB; S2, 66 DAFB; S2-3, 74 DAFB; S3, 90 DAFB; S4, 135 DAFB. PpActin used as reference gene. Error bars are standard error of the mean for three replicate reactions.

Figure 4

Relative expression profiling of biosynthetic genes for branch pathway flavonol and proanthocyanidin synthesis in skin and flesh of ‘Stark Red Gold’ nectarines. The developmental stages are indicated as follows: S1, 50 DAFB; S2, 66 DAFB; S2-3, 74 DAFB; S3, 90 DAFB; S4, 135 DAFB. PpActin used as reference gene. Error bars are the standard error of the mean for three replicate reactions.

Figure 5

Phylogenetic relationships between Arabidopsis MYB transcription factors and polyphenolic-related MYBs of other species. Sequences were aligned using Clustal W (opening = 15, extension = 0.3) in Vector NTI 9.0. Phylogenetic and molecular evolutionary analysis was conducted using MEGA version 3.1 [51] using minimum evolution phylogeny test and 1000 bootstrap replicates.

Figure 6

Relative expression analyses of the transcripts of MYB10, bHLH3 and WD40 in peel and flesh of ‘Stark Red Gold’ nectarines during development and ripening. The developmental stages are indicated as follows: S1, 50 DAFB; S2, 66 DAFB; S2-3, 74 DAFB; S3, 90 DAFB; S4, 135 DAFB. Error bars are the standard error of the mean of three replicate reactions.

Figure 7

Relative expression analyses of the transcripts of the potential MYB flavonoid activators, MYBPA1, MYB15 and MYB123 in peel and flesh of ‘Stark Red Gold’ nectarines during development and ripening. The developmental stages are indicated as follows: S1, 50 DAFB; S2, 66 DAFB; S2-3, 74 DAFB; S3, 90 DAFB; S4, 135 DAFB. Error bars are the standard error of the mean of three replicate reactions.

Figure 8

Relative expression analyses of the transcripts of the potential MYB flavonoid repressors, MYB111 and MYB16 in peel and flesh of ‘Stark Red Gold’ nectarines during development and ripening. The developmental stages are indicated as follows: S1, 50 DAFB; S2, 66 DAFB; S2-3, 74 DAFB; S3, 90 DAFB; S4, 135 DAFB. Error bars are the standard error of the mean of three replicate reactions.

Figure 9

Interaction of the peach promoters (A) LAR, (B) DFR and (C) UFGT by peach MYB10 and peach MYBPA1 with and without peach bHLH3 in a dual luciferase transient tobacco assay. Leaves of N. benthamiana were infiltrated with the peach promoter-LUC fusion on its own (control) or co-infiltrated with MYB10 +/− bHLH3. Luminescence was measured 3 d later and expressed as a ratio of LUC to REN. Data are presented as means (± SE) of six replicate reactions.

Figure 10

‘Stark Red Gold’ nectarines harvested from the most shaded parts of the canopy at harvest time (T0) and after 72 hours kept under UV + white light (T1 exp) or in the dark (T1 cov) (A)., and (B) Expression analyses of the transcripts of DFR, UFGT, MYB10, bHLH3 and WD40 in the peel of the fruit shown in (A). Error bars are the standard error of the mean of three replicate reactions.